The transforming growth factor-beta (TGF-.beta.) superfamily of cytokines regulate a diverse array of physiologic functions including cell proliferation and growth, cell migration, differentiation, development and apoptosis. This large family includes the TGF-.beta.s, activins, and bone-morphogenic proteins (BMPs) and each subgroup initiates a unique signaling cascade activated by ligand-induced serine/threonine kinase receptor complex formation (Wrana, Miner. Electrolyte Metab., 1998, 24, 120-130). These complexes, once formed, recruit and phosphorylate members of a family of cytosolic proteins, known as Smads. Smads exist as monomers in unstimulated cells but homo- or heterodimerize and translocate to the nucleus activating target gene transcription upon ligand binding. Smads, therefore, connect the pathway of TGF-.beta. signaling from the cell membrane to the nucleus.
To date, nine vertebrate Smads have been identified and these have been divided into subgroups based on their functional role in various pathways. Smad1, 5, and MADH6, which is 80% homologous to Smad1, all mediate signal transduction from BMPs while Smad2 and 3 mediate signal transduction from TGF-.beta.s and activins. Collectively, these Smads are known as the pathway-restricted Smads and can form homo or heterodimers. Smad4 has been shown to be a shared hetero-oligomerization partner to the pathway-restricted Smads and is known as the common mediator. The last two members of the family, Smad6 and 7, act to inhibit the Smad signaling cascades often by forming unproductive dimers with other Smads and are therefore classified as antagonistic Smads (Heldin et al., Nature, 1997, 390, 465-471; Kretzschmar and Massague, Curr. Opin. Genet. Dev., 1998, 8, 103-111).
Smad7 (also known as MADH7) is a member of a subgroup of Smad family transcription factors which act to inhibit signaling by other Smad members. Smad7 was isolated from cultured human vascular endothelial cells using a differential display approach as a molecule selectively induced by laminar shear stress (Topper et al., Proc. Natl. Acad. Sci. U.S.A., 1997, 94, 9314-9319). In these studies, Smad7 was capable of modulating endothelial gene expression in response to TGF-.beta. and biomechanical stress. In addition, Smad7 was shown to selectively associate with Smad6, a related antagonistic Smad, and no other Smads. These results suggest that Smad7 may act as an antagonist at the level of Smad-receptor interaction and not by binding and sequestering other Smad members. Recently it was shown that Smad7 interacts with the TGF-.beta. receptor complex and interferes with the phosphorylation of Smad2 and Smad3 (Nakao et al., Nature, 1997, 389, 631-635) as well as the association of Smad2 with Smad4 (Hayashi et al., Cell, 1997, 89, 1165-1173). In addition to TGF-.beta. cascades, Smad7 has also been shown to mediate activin-induced pathways. Ishisaki et al. demonstrated that Smad7 expression suppressed activin induced apoptosis in HS-72 cells (Ishisaki et al., J. Biol. Chem., 1998, 273, 24293-24296).
Regulation of Smad7 activity appears to involve negative feedback mechanisms. Smad7 expression has been shown to be upregulated upon TGF-.beta. and BMP treatment of cells, further illustrating the comprehensive nature of Smad7 antagonism of the Smad signaling cascades (Takase et al., Biochem. Biophys. Res. Commun., 1998, 244, 26-29; Topper et al., Proc. Natl. Acad. Sci. U.S.A., 1997, 94, 9314-9319).
Smad7 mRNA is expressed in all human tissues examined with the highest expression being in the lung. This result led to the investigation of Smad7 expression in various lung cancers. Smad7 was found to be highly expressed in the SCLC cell lines U-1690, H-69 and H-82 and in the non-SCLC cell lines H-157 and H-125 (Afrakhte et al., Biochem. Biophys. Res. Commun., 1998, 249, 505-511). Smad7 demonstrates an endothelial-selective pattern of expression in blood vessels, both normal and atherosclerotic. This Smad family member is therefore believed to be relevant to a variety of physiologic and pathophysiologic processes in the cardiovascular system (Topper et al., Proc. Natl. Acad. Sci. U.S.A., 1997, 94, 9314-9319; Topper et al., J. Clin. Invest., 1997, 99, 2941-2949).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of Smad7. To date, strategies aimed at inhibiting and/or investigating Smad7 function have involved the use of antisense expression vectors.
Transfection of antisense Smad7 expression plasmids into Mv1Lu cells increased the TGF-.beta.1 mediated activation of a promoter containing a luciferase reporter, leading to the conclusion that the expression level of Smad7 determines the cellular response to TGF-.beta.1 (Afrakhte et al., Biochem. Biophys. Res. Commun., 1998, 249, 505-511).
In light of the lack of strategies targeting Smad7 there remains a long felt need for additional agents capable of effectively inhibiting Smad7 function. Therefore, antisense oligonucleotides may provide a promising new pharmaceutical tool for the effective and specific modulation of Smad7 expression.